Process of purification of gland extracts



Patented Mar. 23, 1937 UNITED STATES PROCESS OF PURIFICATION OF GLANDEXTRACTS Wilbur Willis Swingle and Joseph John Pfifiner,

Princeton, N. Company, Michigan 3., assignors to Parke, Detroit, Mich acorporation Davis & o!

No Drawing. Application September 4, 1930, Serial No. 479,802. RenewedMay 1'], 1935 3 Claims.

The invention relates to aprocess for purification of glandular extractsand the products obtained thereby. More particularly the inventionrelates to a purified extract containing the hormone of the suprarenalcortex and the method of obtaining such extract.

We have discovered that it is possible to purify solutions containingthe hormone of the suprarenal cortex by subjecting the solution to atreatment with certain complex silicates such as zeolites of whichpermutit is a typical example. By passing the solution through a filtercontalning permutit a large amount of the foreign material contained inthe solution is removed while the active principle or hormone passesthrough unchanged. The filtrate therefore con- I 'geously tains all ofthe activity of the original solution but in such a purified state thatthe local reaction upon subcutaneous injection of the same is reduced toa negligible amount.

One method of carrying out our invention is to use the 70% alcoholicsolution of the hormone of the suprarenal cortex obtained in accordancewith the directions given in our copending application Serial No.478,558 filed August 28, 1930. In the application referred'to theadrenal glands of cattle are first extracted with ethyl alcohol, thentreated with benzene to remove proteins, inert material and epinephrine.The benzene is removed from the solution and acetone added to removephospholipids. The acetone is then removed and the residue treated withpetroleum ether and aqueous alcohol to separate neutral fat andcholesterol. The result of this treatment is that the hormone isretained in the 70% alcoholic solution and it is from this point thatour improvedffinetho'd of purification is advantarried out.

'I'h; =&ICOhOIiO solution contains 1.49 grams of solids including 36milligrams of epinephrine. The solvents are removed by distillation inpartial vacuo at an external bath temperature of -50 C. Toward. the endof the distillation small quantities (about 30 to cc.) of absolute ethylalcohol are added to facilitate the removal of most of, the water. Theresidue is dissolved in 100 cc. of ethyl alcohol. The solution thusiormed is filtered through permutit in order to remove foreign materialsfrom the solu tion may be repeated the second time.

tion while retainingthe entire activity oi. the hormone.

A convenient method for carrying out this phase of the process consistsin the use of two filtering tubes, one above the other, in each of 5which there is placed a small pledget of cotton and a layer of permutit.The solution is then caused to flow through the filtering tubes at therate of about 1 to 2 drops per second. After the alcoholic solution ofthe hormone has entered 10 the filter the permutit is washed first witha cc. portion of 95% ethyl alcohol and then with a 300cc. portion. Theseare allowed to pass through the filters at about the same rate as thealcoholic solution of the hormone and the flow 15 is controlled so thatthe level of the wash alcohol in the filtering tube is preferably a fewcentimeters above the level of the permutit. A gentle suction isrequired at the end of the washing.

The alcoholic filtrate resulting from the above 2 process contains muchless solids than the original 70% alcoholic solution. If desired thefiltra- The alcoholic solution is concentrated to about 100 cc. and '70cc. of distilled water are added. The 25 amount of water added at thispoint depends on the concentration of extract desired. The alcohol isremoved and the extract diluted to 100 cc. with distilled water. A milkysuspension is obtained which can be very rapidly and con- 39 venientlyclarified and sterilized by passing through a Seitz filter.

The filtered extract pale yellow in color.

is crystal clear and very The finished extract contains less than 0.3gram of solids including 35 approximately 0.05 mg. of epinephrine. Thusthe permutit filtration has removed more than a gram of solidsincluding'35 milligrams of epinephrine while the potency of the extractis as good as the original solution. Bythe addition of sodium chloridethe extract may be rendered isotonic and the solution may bestandardized so that 1 cc. represents 30 grams of fresh cortex. Thepermutit used in the process outlined above can be reclaimed by washingit thoroughly withconcentrated sodium chloride solution, then withdistilled water and dried. While we have indicated above one method ofprocedure for carrying out our invention, it is to be clearly understoodthat the invention in its 2 aovacce broader aspects is capable of otherembodiments. What we claim as our invention is:- We have discovered thatany solution containing 1. The method of purifying an extract of the theactive principle of the suprarenal cortex may suprarenal glandcontaining the cortical horbe passed through permutit without loss ofpomone comprising dissolving material from the tency. For example any ofthe following solvents cortical portion of the suprarenal gland in a forsuprarenal cortex hormone may be used: solvent capable of preserving theactivity there- Ether, petroleum-ether, other alcohols such as of,separating therefrom proteins, inert material, propyl alcohol and butylalcohol, acetone, water, epinephrine, phospholipids, neutral fat andetc. 4 cholesterol, dissolving the cortical hormone ob- Thus thefiltration through zeolites may if detained from the previous steps in70% alcoholic sired be carried out earlier in the sequence of solution,distilling said solution in partial vacuo steps given in our co-penclingapplication' to remove solvents and water, dissolving the In the abovedescription the term permutit residue in 95% ethyl alcohol, andfiltering said is used to designate a ,well known material usedalcoholic solution through permutit thereby ob-= primarily for watersoftening processes. Our intaining the entire activity of the corticalhorvention, however, is not limited to this exact mamone free fromepinephrine. terial but may be carried out by using other sim- 2. In theprocess of purifying an extract of ilar materials of the type known asbase exchange the suprarenal cortex containing the cortical materials.Complex silicates such as zeolites or hormone the step of filtering asolution containother materials which are capable of causing an ing thecortical hormone and epinephrine through exchange of bases can be used.permutit and obtaining in the filtrate the corti- It is probable thatthis material actsto recal hormone free from epinephrine without lossmove certain organic bases which are present in of cortical hormoneactivity. the extracts obtained from the suprarenal gland 3.-In theprocess of purifying an extract of the and to substitute inorganic basestherefor. The suprarenal cortex containing the cortical horremoval ofsuch organic bases results in a purifimone, the step of filtering asolution containing cation of the extract to a degree that it can be thecortical hormone and epinephrine through injected subcutaneously inlarge amounts withzeolite thereby obtaining in the filtrate the coroutcausing local irritation at the site of injectical hormone free fromepinephrine without loss tion. Therefore our invention results inobtainof cortical hormone activity.

ing a highly purified extract containing the cortical hormone and makesavailable a therapeutic WILBUR WILLIS SWINGLE. product of great value.This product is suitable JOSEPH'JOHN PFIFFNER. for subcutaneous,intraperitoneal, and intravenous use in man and animals. I

